title: Metabarcoding for NoV surveillance: undetermined sample sequencing
description: Norovirus (NoV) specific metabarcoding was performed employing Illumina short-read sequencing. This amplicon-based approach takes advantage of the well-established primers used for NoV genotyping, that target specific, well-studied regions of the viral genome, namely portions of the genes encoding the RNA-dependent RNA polymerase (RdRp) and the capsid protein (VP1) (Imamura et al., 2017, Lun et al., 2018, Oshiki et al., 2018). In addition, primers that target a longer fragment (400-500 nt) comprising a part of RdRp and a part of VP1 gene sequences were also employed, thus allowing the typing of both genes in the same amplicon (dual typing). The PCR amplicons were tagged and indexed by an additional PCR, purified, quantified and mixed into a pool to perform sequencing on the Illumina Miseq platform. The raw reads, are thus, amplified genome fragment products that arise from different (Noro)viruses present in the sample. Raw reads were demultiplexed directly by the Illumina MiSeq sequencer. Reads quality was verified using FastQC (V.0.11.8 https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Andrew, 2010) and MultiQC (V.1.9 https://multiqc.info/ Ewels et al., 2010) tools. Reads were assembled into clusters using the FROGS suite in a Galaxy environment (Escudié et al, 2018). The sequence of each cluster was genotyped using the Norovirus Typing Tool 2.0 to allow for the characterization of the diversity of the NoV genotypes. Samples come from two different sequencing sets - Baseline Survey (BLS) samples, and two synthetic Test Set samples (TSS1 and TSS2).